Compositions and methods for pairwise sequencing

A method for pairwise sequencing, comprising:

• a) providing a plurality of immobilized nucleic acid concatemer template molecules, wherein individual nucleic acid concatemer template molecules comprise at least one uridine that can be enzymatically cleaved to generate an abasic site in the concatemer template molecule,
• wherein individual nucleic acid concatemer template molecules in the plurality are immobilized to a first surface primer that is immobilized to a support, and
• wherein individual first surface primers in the plurality lack nucleotides having a scissile moiety;
• b) sequencing the plurality of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers, a plurality of sequencing polymerases, a plurality of multivalent molecules and a plurality of nucleotide analogs, thereby generating a plurality of extended forward sequencing primer strands, wherein individual immobilized concatemer template molecules have two or more extended forward sequencing primer strands hybridized thereon;
• c) retaining the plurality of immobilized concatemer template molecules and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands that are hybridized to the retained immobilized single stranded nucleic acid concatemer template molecules by conducting a primer extension reaction;
• d) removing the retained immobilized concatemer template molecules by generating abasic sites at the at least one uridine in the immobilized concatemer template molecules and generating gaps at the abasic sites to generate a plurality of gap-containing concatemer template molecules while retaining the plurality of forward extension strands and retaining the plurality of immobilized surface primers;
• and
• e) sequencing the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers, a plurality of sequencing polymerases, a plurality of multivalent molecules and a plurality of nucleotide analogs, thereby generating a plurality of extended reverse sequencing primer strands, wherein individual retained forward extension strands have two or more extended reverse sequencing primer strands hybridized thereon,
• wherein individual multivalent molecules of the plurality of multivalent molecules comprises a core attached to a plurality of nucleotide arms via their core attachment moiety, the nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit.